The quality of tissue dissociation enzymes (TDEs) containing collagenase was critical to the success of the Edmonton Protocol, an islet transplantation procedure that led to successful treatment of adult type 1 diabetic patients who were disabled by refractory hypoglycemia. The quality and yield of human islets recovered from pancreata were improved when Liberase(tm) HI Purified Enzyme Blend (HI) was used in place of crude collagenase to dissociate the tissue. HI has become the primary TDE reagent for human islet isolation because of its defined compostion (contains 3 purifed proteases: two classes of collagenase -class I and class II- purified from Clostridium histolyticum culture supernatants and thermolysin, a bacterial neutral protease) and low amounts of endotoxin contamination. The number of islet transplants performed over the last 6 years (~550) since publication of the Edmonton Protocol now exceeds the number of transplants performed the prior 26 years (n=483). A strong concern of the islet transplant community is inconsistent islet yields that many believe reflects the variable quality of the HI product (see http://icr.coh.org/workshops.asp). Collagenase is the critical reagent since it is the only enzyme that can degrade collagen fibers present in the extracellular matrix. Once the fibers are cut, other neutral proteases accelerate the degradation of the collagen leading to release of islets from the tissue. Inconsistent islet yields may in part be due to the protease degradation of collagenase during the protein purification or tissue dissociation process. Structure-function studies of these enzymes showed that once degraded, they are ineffective in cutting collagen. This issue will not be resolved until a superior purifed collagenase enzyme is used in purified TDE products. This project addresses this issue by developing a protease resistant, recombinant (rec) class I & class II enzymes using sequences of C. histolyticum genes found in public databases. Once this reagent is developed, then systematic studies can be performed to assess factors that affect the quality and yield of islets. The basic elements of the project are as follows: CI or CII collagenase purified from C. histolyticum culture supernatants will be digested with neutral proteases present in the tissue dissociation mixture, the peptides purified by reverse phase HPLC, then the sensitive residues determined using mass spectrometry. Critical amino acid residues attacked by proteases will be changed by using PCR mutagenesis techniques and the mutated genes used to express the rec enzymes. After confirming the resistance of the mutated collagenases to protease degradation, enzyme production will be scaled up, & the mutant enzymes purified and assessed for their effectiveness to isolate human and porcine islets. Collagenase is a critical reagent for isolating islets from pancreatic tissue. The success of islet transplantation as a cellular therapy to treat adult type diabetic patients relies on isolating a sufficient number of high quality islets from human pancreata. The inconsistent quality of purified collagenase used in islet isolation procedures today often leads to suboptimal islet yields. Development of recombinant collagenase enzymes that are resistant to protease degradation will minimize the "collagenase problem" and begin to build a foundation for obtaining a deeper understanding of other factors that affect islet yield. [unreadable] [unreadable] [unreadable]